Humans can form vitamin D3 (cholecalciferol) in the skin using the UV portion contained in sunlight. Vitamin D2 (ergocalciferol) is taken up from food. Ergo- and cholecalciferol differ in their side chains and biological activity. However, they are both bound by the vitamin D binding protein (VDBP) in circulation and metabolized to 25-hydroxyvitamin D (25(OH)D or calcidiol) in the liver (see Schmidt-Gayk H, et al (ed.), Calcium regulation hormones, vitamin D metabolites and cyclic AMP, Springer publishing, Heidelberg (1990), pages 24-47). 25(OH)D represents the storage form in the body and is the vitamin D metabolite with the highest concentration in blood. When needed, 25(OH)D becomes hydroxylated in the kidney by 25-hydroxyvitamin D-1α-hydroxylase to 1α,25-dihydroxyvitamin D which is the D-hormone or calcitriol and the biologically active form. The activity of the vitamin D-hydroxylase is multiply regulated, e.g. by the parathyroid hormone and by the calcium level in the blood. Calcitriol is bound by the vitamin D receptor (VDR) intracellularly and transported into the nucleus where the complex with the vitamin D receptor associates to the DNA, leading to changes in protein synthesis.
Determination of vitamin D metabolites in blood or serum is medically indicated in case of suspected vitamin D deficiency, for example, due to insufficient synthesis, reduced intestinal absorption and malabsorption, liver dysfunction, increased vitamin D metabolism following the intake of antiepileptic drugs, increased loss of vitamin D in case of a nephrotic syndrome or a general disturbance of the calcium-phosphate balance. Up to one third of the elder normal population in Northern Europe are suffering for example from vitamin D deficiency when sunlight is poor during winter times (Sharla et al., Osteoporosis Int 1998; 8(2):7-12). A slight deficiency of less than 15 nanograms calcidiol per milliliter serum (37.5 nmol/L 25-OH-Vit. D/L) causes a rise in parathormone levels and increased bone resorption due to decreased calcium intake (Chapuy M C et al., J Clin Endocrinol Metab 1996, 81:1129-33). Vitamin D deficiency is therefore an important risk factor for senile osteoporosis. A severe deficiency of less than 5 nanograms calcidiol per milliliter serum (12.5 nmol/L) causes rachitis in children and osteomalacia in adults (Sharla et al., Exp Clin Endocrinol Diabetes, 1996, 104:289-292). A vitamin D deficiency is associated with a higher risk of breast, colon and prostate cancer and various autoimmune diseases such as juvenile diabetes and multiple sclerosis (Holick M F, Vitamin D deficiency. N Engl J Med (2007) 357:266-81). A vitamin D overdose causes the hypocalcaemia syndrome. The minimum serum concentration for bone health lies between 20 to 32 ng 25(OH)D/mL (50-80 nmol/L) and a concentration of more than 30 ng/mL or rather 75 nmol/L 25(OH)D should be sought.
The chemical methods for determination of vitamin D metabolites are laborious (see Tanner et al, J. Assoc of Analyt Chem (1988) 17, 607-710; Haddad, J G et al., J Clin Endocrinol Metab (1971) 33, 992-995 and Eisman J A et al., Anal Biochem (1977) 80, 298-305). U.S. Pat. No. 5,981,779 (Holick et al.), WO 89/01631 and EP 0 583 945 (DeLuca et al.) teach immunological determination methods. Prior to immunological determination, samples must be carefully prepared since more than 85% of vitamin D metabolites in human serum are bound to VDBP, albumin and other proteins. WO 99/67211 (Armbruster et al.) teaches an ethanol precipitation of the serum proteins and an analysis of vitamin D metabolites in the ethanol supernatant. EP 0 753 743 (Hollis) recommends a periodate precipitation of serum proteins and an analysis of the protein-free aqueous supernatant. DE 10 144 905 C2 (Armbruster et al.) teaches an analysis directly in serum following a release of the vitamin D metabolites from their binding sites through the addition of displacement agents such as salicylic acid, warfarin or aniline sulfonic acid. WO 2002/057797 (Quest Diagnostics Inc.) claims the addition of salicylic acid, cyclodextrin and a change of pH to obtain a release of vitamin D metabolites. EP 2 126 586 B1 (Immundiagnostik AG) teaches the digestion of serum proteins by a serine protease followed by an immunoassay with a protease-insensitive monoclonal antibody. In various automated procedures, the serum sample is subjected to deproteinization and delipidation using acetonitrile. WO 2011/122948 (Future Diagnostics BV) discloses a release of vitamin D metabolites in serum samples using a fluorine-containing surfactant such as perfluorooctanoic acid.
U.S. Pat. No. 7,745,226 B2 (Clarke et al.) teaches a combination of liquid chromatography and mass spectrometry for determination of vitamin D metabolites in serum. The various LC-MS, LC-SMS and LC-TMS are hereinafter referred to as LC-MS methods (see Vogeser M, Liquid chromatography-tandem mass spectrometry•Application in the clinical laboratory, Clin Chem Lab Med (2003) 41:117-26; Vogeser M. Seger C. A decade of HPLC-MS/MS in the routine clinical laboratory—goals for further developments. Clin Biochem (2008) 41:649-62). While LC-MS analysis is considered the new standard in vitamin D analysis, there are controversies on the reliability and interpretation of the measured values because, historically, the reference values had been established by immunological methods and all therapeutic recommendations are based on them. The immunological methods usually show about 15 percent lower values than the LC-MS methods. A conversion of the values is not possible, since many of the 25(OH)D levels in serum measured by means of LC-MS in the period from 2007 to 2008 has proven as being too high (see Andrew Pollack, New York Times, 7/8 Jan. 2009). Studies using the Roche automated 25(OH)D assay show that 25-hydroxyvitamin D3 in blood is stable up to 3 days at room temperature; in serum even longer (Wielders et al, Clin Chem (2009) 55(8), 1584-5). The Roche 25(OH)D assay, however, does not detect medication with vitamin D2 and said studies on temperature stability of 25(OH)D3 in serum do not take account of the real pre-analytical events in doctors' offices and hospitals. The 25(OH)D-EIA of Immunodiagnostic System Ltd and its automated version (Siemens ADVIA Centaur Vitamin D Total assay) are also prone to systematic errors (see Cavalier et al., JBMR (2011) 26:434-436). The current LC-MS and immunological methods may therefore lead to false measurements and to a wrong medication, in particular, when hemolysis has taken place in blood or serum samples. The prior art therefore represents a problem.